Ten steps to improve confidence and ensure reproducibility and reliability in managing these experiments.

Hydrogen deuterium exchange mass spectrometry (HDX MS) is gaining recognition for is influential place within the analytical toolbox for the insights it provides to biotherapeutic characterization. HDX MS is used to study a protein’s structural dynamics and conformational changes, a component of understanding a protein’s higher order structure.

Information about protein conformation from an HDX MS study can be used to compare a control with an analyte by measuring the relative amount of deuteration uptake (mass increase) as a function of time. HDX MS is capable of monitoring domain interaction, localized protein breathing, and folding or unfolding in the solution phase.

An HDX MS experiment can be broken into three main stages: deuterium labeling, mass analysis, and data processing. Detailed here are the steps involved in executing HDX MS analysis using the Waters nanoACQUITY UPLC System with HDX Technology, which is designed to facilitate an HDX MS protocol to improve confidence and ensure reproducibility and reliability in managing these experiments.

Preparation: Prepare the protein samples in 10 mM phosphate buffer in H2O (undeuterated) at pH 7.0 (or other buffer conditions that work for proteins at native condition).

Online digestion: The quenched solution is directly injected into Waters HDX Manager for online pepsin digestion. The online pepsin digestion is performed at 20°C at a flow rate of 100 µL/min, and the digested peptides (labeled and quenched) are trapped for 3 min at 0°C using an ACQUITY UPLC BEH C18 1.7-μm, 2.1 x 5 mm VanGuard pre-column.

Liquid chromatography: The peptides are eluted from the trapping column to the analytical column for rapid UPLC separation. The peptides are separated at 0°C in 6 min using an ACQUITY UPLC BEH C18 1.7-μm, 1.0 x 100 mm column at a flow rate of 40 μL/min.

Mass spectrometry: For undeuterated control, the mass of the parent and its fragment ions are measured by MSE, a technology available on Waters mass spectrometers whereby exact mass information is collected at both low and high energy, to identify peptide sequence. The identified peptides are used to construct the coverage map of the protein. For deuterated samples, the masses of the labeled peptides are measured.

Data processing: LC/MS data for undeuterated samples are processed in ProteinLynx Global Server (PLGS) and deuterated samples are processed in DynamX Software, both available from Waters. DynamX is dedicated HDX software that automatically calculates the deuterium uptakes of each peptide from undeuterated to deuterated time-points. The calculated uptakes are displayed in uptake charts, difference plots, and heat maps in DynamX. The HDX data can be combined with 3D structure of protein to display in-depth information about location and interaction. Automated data processing significantly reduces the experimental time from weeks to days.

Comparison: Repeat steps 1 to 7 for multiple sets of samples. For example, the HDX data of a mutated protein can be compared to the HDX data of a reference protein performed under the same analytical conditions. Locate the peptides showing different amounts of deuterium uptake between the reference and mutated proteins. This comparison can be directly related to the difference in localized conformation upon mutation.

Cautionary note: pH 2.5 and temperature control at 0°C are critical parameters in HDX experiments. These conditions should be maintained to prevent a back-exchange phenomenon, which is when labeled deuterium exchanges back to hydrogen, therefore rendering deuterated information meaningless.

Joomi Ahn, Ph.D, is a principal scientist in the Pharmaceutical & Life Sciences Group at Waters Corporation.

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